Preliminary Study On Functions Of MrHal3 Gene In Monascus Ruber M7

Monascus spp.has been receiving great attention from many scientists because they have a vital role in food and medicine fields.In food aspect,Monascus spp.have been used to make rice wine,fermented soybean curd,vinegar and so on in many Asian countries,especially in China and Japan for centuries.Their pigments have been used as colorant agents for fermented soybean curd,yogurt,meat and fish products,ketchup,red wines,and other products.Now,many studies have shown that red fermented rice(RMR)by Monascus spp.contains several types of useful substances.For example,monacolins K,also known as lovastatin which is used as an anti-hyper cholesterolemic agent,Monascus pigments(MPs)which have being usually applied as the natural food colorants,gamma-aminobutyric acid which can be utilized as a hypotensive agent,dimerumic acid which may be utilized as a natural antioxidant,and others.Hal3(halotolerant),also known as Sis2(Sit4 suppressor)is a highly conserved flavoprotein,which widely exists in yeasts,plants and animals,and it has been originally identified as a negative regulatory subunit of Ppz1 protein which plays a role in the regulation of monovalent cation homeostasis,and impacts on cell-cycle control,halotolerance and cell integrity.Recently,researchers have discovered that Hal3 has an important function on growth under blue light and it has been described as a novel pathway of light-modulated for plant growth.The purpose of our research is to investigate the function of MrHal3 in M.ruber M7(Hal3in M.ruber M7)by the analyses of the corresponding gene deletion mutant.In this study,we constructed the MrHal3 deletion mutants successfully.Then,the colonial and microscopic morphologies,colonial growth rate,biomass and the MPs production in darkness,under Na Cl,glycerol and blue light stresses were investigated.The results are described as follows.1.The construction of ΔMrHal3 deletion mutant of M.ruber M7MrHal3 deletion cassette was constructed and linked with p MD19-T vector-mediated and transferred to p CAMBIA3300 then the gene deletion vector which carried MrHal3 cassette was inserted into M.ruber M7 by Agrobacterium tumefaciens-mediated transformation method.Finally,the ΔMrHal3 was verified by the PCR and Southern blot.2.The analyses of phenotypic and pigments production of ΔMrHal3The colonial and microscopic morphologies of M.ruber M7 and ΔMrHal3 cultured on PDA,PDA with Na Cl,G25 N and CYA media were analyzed.The results have showed that on PDA,ΔMrHal3 and M.ruber M7 possess the same colonial sizes,color,and aerial hyphae;on CYA and G25 N,there is no difference of the colonial sizes,but the colonial color of ΔMrHal3 is darker on G25 N and lighter on CYA than of that M7;on PDA medium with high concentrations of Na Cl,the colonial sizes and color of ΔMrHal3 mutant are darker and bigger than M.ruber M7.On PDA,G25 N and CYA,the differences of the microscopic morphologies of ΔMrHal3 and M.ruber M7 have not been seen,and on PDA medium supplied with Na Cl,the differences of the microscopic morphologies of ΔMrHal3 and M.ruber M7 have not been seen.The MPs analysis of ΔMrHal3 was found significant differences,compared with M.ruber M7 under Na Cl,glycerol and blue light stresses.The MPs production significantly decreased under blue light in the strain lack to MrHal3 gene and increased under salt stress;on other hands the biomass of ΔMrHal3 increased under salt stress,500 lux of blue light,and decreased under 900 lux of blue light.Therefore,ΔMrHal3 from M.ruber M7 may have a role in growth and MPs production of M.ruber M7 under Na Cl,glycerol and blue light stresses.